Vpr mediates HIV-1–induced PCS. (A) A schematic structure of HIV-1 pNL43 (original clone) and pNL-LUC-E⁻ (Δenv/wt). wt and mutant viruses were env deficient (Δenv), and nef was replaced by the luciferase (Luc) gene. The accessory genes vif, vpr, and vpu were mutated (yielding Δvif, Δvpr, and Δvpu, respectively). (B) Representative chromosome spreads from PBLs infected or not infected with wt or mutant virus (Δvpr, Δvpu, or Δvif). PBLs were synchronized with colcemid. Cont, control. Bar, 10 µm. (C) A luciferase assay was used to determine the virus production rate (right) and PCS frequency (left). (D) Chromosome spreads were Giemsa stained. At 48 h, the frequency of PCS was >40% in DOX-induced Vpr-transfected cells (far right) and <3% in nontreated cells (middle right). In contrast, mock-transfected cells (ΔVpr) showed no difference in PCS frequency with or without DOX treatment (<3%; left). Bar, 5 µm. (E) The cell cycle was analyzed using FACS after DOX addition (days 0–5). ΔVpr (left) and MIT-23 (right) cells are shown. Arrow, hyperploidy/aneuploidy. All data are representative of at least three independent experiments.